Project Summary/Abstract One crucial aspect of tissue homeostasis is the ability of adult stem cells to self-renew over long periods of time. It is well understood that tissue-specific transcription factors and microRNAs can act as key regulators of stem cell self-renewal and differentiation. Recently, a new class of molecules called long noncoding RNAs (lncRNAs) has been described, and lncRNAs have now been shown to play important biological roles in a variety of cell types. Aberrant lncRNA expression may contribute to human neurological disorders such as schizophrenia, Alzheimer?s disease, and autism. However, it remains poorly understood whether lncRNAs can regulate stem cell self-renewal. The ventricular-subventricular zone (V-SVZ) of the mouse brain contains a population of neural stem cells (NSCs) that can produce neurons as well as glial cells. An essential feature of these NSCs is their ability to self-renew, thus enabling the maintenance of a population of NSCs throughout life to support continued neurogenesis. We have previously identified the lncRNA Pnky as a neural-specific transcript expressed in the neurogenic lineage of the V-SVZ. It was determined that knockdown of Pnky in V- SVZ cultures results in a significant increase in neuroblast production, but the role of Pnky in the V-SVZ in vivo has not yet been explored. Since in vivo lncRNA studies are currently very limited, particularly for neural- specific lncRNAs, the major goal of this work is to analyze the expression and function of the lncRNA Pnky in the V-SVZ in vivo. To this end, a conditional Pnky knockout (Pnky-cKO) mouse has been generated. Preliminary studies of Pnky-cKO mice revealed an initial increase in neuroblast production. However, at later ages neurogenesis was substantially decreased, suggestive of possible stem cell depletion. Therefore, work proposed in Aim 1 will characterize the expression of Pnky within the neurogenic lineage of the V-SVZ. The Pnky-cKO mouse will then be used to analyze the effects of deleting Pnky from V-SVZ NSCs. In particular, whether Pnky regulates NSC quiescence will be tested. Aim 2 will analyze the molecular mechanism through which Pnky functions. It was previously determined that Pnky interacts with the splicing regulator PTBP1. Therefore, Aim 2 will address transcriptome-wide changes in NSCs following Pnky deletion, with particular focus on changes in abundance or splicing of known PTBP1 targets. Together, these aims will test the hypothesis that Pnky is required for the long-term maintenance of V-SVZ NSCs. This analysis of the role of a specific lncRNA in neural stem cells in vivo will contribute novel insights into how lncRNAs can regulate stem cells and underlie disease. The feasibility of this work is supported by the Preliminary Data, strong mentoring team, and customized training plan.